Project Summary Following stromal wounds, intra-stromal cells (resident and bone marrow-derived) change or `transform' into myofibroblasts (MFs). This change involves the synthesis ?-SMA containing stress fibers and secretion of extracellular matrix components. Persistence of the myofibroblast phenotype brings about fibrosis, i.e., the formation of dense, disorganized opaque collagenous material that blocks and distorts vision. MF genesis is controlled by TGF?. Exposure of the mouse eye to alkali induces the de novo expression of ?-SMA and complete corneal opacification. Japanese researcher demonstrated that these events do not occur in Transient Receptor Protein Voltage activated channel one knockout mice (TRPV1-/- mice), indicating a critical role of this channel in the fibrotic process. Following their groundbreaking studies, a collaborative study in corneal fibroblasts between three laboratories demonstrated that, a) myofibroblast formation in the wounded pig cornea is also dependent on TRPV1 activity; b) the phenotype change is underpinned by a positive feedback process that starts when the activated TGF? receptor induces SMAD2 activation and the concurrent generation of ROS through a process that is NADPH oxidase (NOX)-dependent; c) in turn, ROS activates TRPV1 leading to a [Ca] increased) this [Ca] rise is instrumental in the activation of p38 (p-p38), and d) in turn, p-p38 directly or indirectly increases pSMAD2 levels establishing thereby recurrent cycles of .pSMAD2->ROS->TRPV1->p- p38->pSMAD2 soon after TGF?R activation. This recurring positive feedback loop is essential to generate the accumulation of the high levels of pSMAD that are necessary to drive maximal fibroblast to myofibroblast conversion (FMC). We seek now to identify and characterize the full complement of proteins and transduction events that underpin the described cycle. In Specific Aim 1 using genetically encoded fluorescent sensors we determine the temporal relationships between the start of ROS generation, pSMAD2 activity and p38 phosphorylation and identify the NOX subtype(s) involved and it (their) location(s). In Specific Aim 2, we address the involvement of intracellular kinases of the MAPK cascades upstream from p38 and test a novel hypothesis for the mechanisms involved in the p.p38 enhancements of SMAD2 activation and use phosphor- proteomic approaches to identify undiscovered mediators of the feedback loop. In Specific Aim 3, we test the hypothesis that the cell culture results are accurate predictors of outcome in an organ culture pig model that appears to be relevant to physical and chemically induced human corneal fibrosis and test the impact of NOX4 in corneal fibrosis in NOX4-/- mice. The spatial and temporal information gathered in the studies above on ROS production, protein phosphorylation changes and other activated entities that will be critical to elucidate the sequence of signal transduction cause and effect in the induction of the myofibroblast phenotype from corneal keratocytes.